Chemoprevention has the potential to be a major component of the control of colon cancer, one of the commonest malignancies. Although traditional non-steroidal anti-inflammatory drugs (NSAIDs) prevent colon cancer, their side effects are a major obstacle to their large-scale application to prevent colon cancer. The novel nitric oxide (NO)-releasing NSAIDs (NO-NSAIDs), consisting of a NSAID molecule and a NO-releasing moiety linked to it via a chemical spacer, have been synthesized to overcome NSAID toxicity. Current evidence suggests that they are much safer than traditional NSAIDs. Early studies indicate that an NO-aspirin (NO-ASA) derivative is much more effective than ASA in cultured cells and against colon preneoplastic lesions. That NO-ASAs have superior effectiveness and safety makes them promising chemopreventive agents and constitutes a compelling argument to study their mechanism of action in colon carcinogenesis. The proposed studies will evaluate two hypotheses: a) NO-ASA compounds share with traditional NSAIDs some of their known mechanisms of action, such as their effects on cell kinetics and on the eicosanoid and NO pathways, all relevant to chemoprevention, and b) Since NO-ASA derivatives are markedly more effective than unmodified ASA, the additions to the ASA molecule to generate NO-ASA impart on it new properties, which make it more effective. Our specific aims are 1) Determine in cultured colon cancer cells the effect of NO-ASA derivatives on cell kinetics (proliferation, apoptosis) and cell cycle; on the eicosanoid pathway (effect on the catalytic activity of COX; COX-1 and -2 expression and regulation; evidence for effects beyond COX); and on the nitric oxide pathway (expression of NOS isoforms; NOS catalytic activity). 2) Determine in an animal model of colon cancer the contribution of the NO-ASA-induced changes to colon carcinogenesis. Based on results from Specific Aim #1, we will select the most promising of the three NO-ASA derivatives and assess its chemopreventive efficacy against azoxymethane-induced colon carcinogenesis in F344 rats. The effect of this compound on cell kinetics and on the two metabolic pathways will be assessed and correlated with chemopreventive efficacy. 3) Assess the contribution of the key structural components of the NO-ASA molecule to their chemopreventive actions. We will study the effects of synthetic molecules representing the structural components of each one of the three NO-ASA compounds. They include: ASA+spacer (NO- ASA without the -NO2 group) to test the contribution of NO; spacer (the part linking ASA to -NO2) to test its individual contribution; and a NO-donor which releases NO, to further test the role of NO. The long-term goal of these studies is to develop mechanism-driven safe and effective strategies for colon cancer prevention.